Posts from Behind the Bench @ Thermo Fisher Scientific

When it comes to quantifying molecular targets in chronic myeloid leukemia (CML) samples, real-time PCR has long been trusted as the gold standard. However, new research is showing that digital PCR holds new promise in two key areas: increased sensitivity and absolute quantification.

As Mary Alikian from Imperial College London explains in the latest LabChat, real-time PCR presents the challenge of quantification using standard curves, which always made it difficult for different labs to compare their data. On top of eliminating the need for standard curves, digital PCR also allows for deeper quantification amidst a background of wild types. According to Mary, quantification can feasibly increase from 1 in a 100,000 to 1 in a 1,000,000 using digital PCR.

Which master mix do you choose? How do you know it’s the right one? Choosing the right master mix is an important step when performing your experiment because you want to make sure it is completely optimized for success.

It’s a constant argument. There have been rap battles that tear labs apart… Which do you use, SYBR® or TaqMan® master mix?

So what is a master mix anyway? It’s a mixture of polymerase, salts, magnesium, dNTPs, and optimized reaction buffer. Your template and primers are the only additions necessary to perform qPCR. Different formulations are good for different situations or applications such as Genotyping,

After his presentation at ESHG 2015, Dr. Csaba Bödör, Semmelweis University, shared his experience as an early access customer for the new Applied Biosystems QuantStudio™ 3 and 5 Real time PCR systems.

Dr. Bödör uses Competitive Allele-Specific TaqMan® PCR (or castPCR) to detect and quantitate rare mutations in blood in leukemia and other myeloproliferative disorders and hopes that the research he is doing will ultimately enable development of better diagnostics in the future. Dr. Bödör found the new QuantStudio instruments to be very user friendly and is excited about the new connectivity and data analysis that is possible with these cloud enabled instruments.

Do you ever find yourself asking - "How does fragment analysis work and why would one perform fragment analysis in their lab?"

Let's check it out.

Fragment analysis refers to a genetic analysis technique used for a wide variety of applications such as mutation detection, genotyping, DNA profiling, genetic mapping and linkage analysis. Various diseases, conditions and chromosomal abnormalities are detected by this method.

Whether you are new to next generation sequencing, or an old hand, you have likely encountered terms like base quality, Q-score, consensus accuracy and so on. This could have happened when you were reading a NGS review or listening to a bright-eyed grad student presenting her new variant calling algorithm at a poster session. Either way, you may have been left thinking, “Huh?”

Let’s turn that “Huh?” to an “Oh, yeah” while we take a closer look at the statistics of sequencing.

We at Behind the Bench hope you have enjoyed this series on Sanger Sequencing by Capillary Electrophoresis, and we conclude with this final post to point out different online resources to access.

There are three guides available to download. The first, the Fragment Analysis Guide, is a 218-page electronic PDF comprehensive in its scope. This guide covers topics from experimental design (including different dye and size standards and primer design considerations), to optimization of PCR, to optimization of capillary electrophoresis for fragment analysis (at instrument-specific detail).

As next generation sequencing evolves, the ability to create reproducible, high quality libraries becomes increasingly important. The Ion AmpliSeq™ method has proven useful to reliably generate quality libraries from as little as 10 ng of nucleic acid. We developed the Ion Chef™ system to offer an automated solution for templating and sequencing chip preparation. Here we demonstrate the marriage of Ion AmpliSeq reagents with the Ion Chef system to create a fully automated library preparation solution.

As a researcher, you’ve been given many choices in terms of tools and techniques. And for some who are intimately familiar with real-time PCR, you are probably hearing about digital PCR and its emerging applications. So the question is, when do you use one or the other or both.

Let’s go back to the basics and explore the technology platform that has been regarded as the gold standard for many years. You guessed it – we’re talking about Sanger Sequencing by capillary electrophoresis. Many might ask, “why is it called Sanger Sequencing?” Sanger Sequencing is named after the inventor of this ground breaking technology, Dr. Frederick Sanger, who developed this method over 40 years ago in the mid-70s. So, what are the basics of Sanger Sequencing?

On-demand video is part of everyday life. Streaming services let you binge watch your favorite TV show or even learn how to batheyour hedgehog. It also turns out that researchers are turning to YouTube to find answers to their everyday questions in the lab. Thermo Fisher Scientific figured this our pretty early on, and launched a dedicated video series for qPCR, called Ask TaqMan. Since premiering in June 2012, the Ask TaqMan has tallied up over a quarter million views with over 9800 hours of viewing time by life science researchers. What’s more, the videos are based on real-world questions sent in by viewers.

Detection and quantification of mutant alleles in tumor tissue is important to cancer research. Testing for the presence of mutations incirculating free DNA (cfDNA) is one of the less invasive research methods available at this time. Digital PCR presents a research tool for mutation detection in cfDNA at a sensitivity level of 1% and below. Challenges associated with digital PCR experiments for rare allele detection include understanding the limit of detection of the assay and platform. This work compares false positive assessment strategies using the signal levels of the no-amplification cluster. Once the false positive call rate is established, this work outlines a method to determine the limit of detection of the assay and platform, at a given level of confidence. Given the number of partitions, the interrogated volume and the false call rate, the tradeoffs between sample load and sensitivity are also discussed.

Just moments before presenting his poster at ESHG in Glasgow, Edgar chats with us about the gold standard for mutational analysis, Sanger Sequencing, a few new tools for analysis, and his poster on the new Minor Variant Finder Software tool.

At ESHG 2015 in Glasgow, it wasn't too hard for attendees to have their heads in the clouds - see what I did there? And when it comes to dealing with your projects' data, that's a good thing! Mike Lelivelt discusses the advances that have been made on the Thermo Fisher Cloud in order to extend the user experience into the Cloud. Data is able to be stored, managed, shared with collaborators, accessed from either home or work and no software needs to be installed for analysis because the tools are already there. And since all the data is managed online, you don’t have to worry about keeping large files locally, where they can take up computing space and may be more susceptible to loss.

Survey and interview studies conducted over a three year period revealed that researchers are not satisfied with their current reversetranscriptase and are performing reactions with i

ncreasingly difficult samples, such as poorly purified RNA and unpurified RNA (direct RT) that both contain inhibitors. To meet this performance gap, the Thermo Fisher Life Sciences Solutions group produced a new reverse transcriptase, SuperScript® IV, and experiments we performed show that it is the most robust reverse transcriptase compared to other enzymes. SuperScript® IV characterization was performed in the context of “real world” situations where users do not have perfect RNA samples. In the presence of a variety of inhibitors, we demonstrate that SuperScript® IV possesses superior performance in a variety of inhibitors, such as alcohols, salts, detergents, phenol, heparin, hematin, bile salts, and formalin typically found in sample preparation reagents, cell lines, blood, feces, and FFPE samples. This enzyme can even detect RNA targets in unpurified RNA samples (directly lysed cells) and whole blood without sacrificing sensitivity and yield. The introduction of SuperScript® IV enables researchers to obtain more consistent results independent of sample quality and simplify and speed up workflows by eliminating RNA purification. -

Kamini Varma discusses her Poster on Using Sanger Sequencing for orthogonal confirmation

of NGS data. The introduction of defined Ion AmpliSeq™ panels for detection and characterization of actionable mutations occurring in tumor tissue has the potential to revolutionize translational oncology research. The Ion Ampliseq™ cancer hot spot panel version 2 (CHP v2) by Ion Torrent includes 207 actionable Direct Sanger CE Sequencing of Individual Ampliseq Cancer Panel Targets from Limited Amount of Input DNA using the Original Ion AmpliSeq™ Pre-Amplification Library. We want to provide a reflex solution for verifying and following up NGS results by Sanger sequencing particularly for samples with VERY limited amount of available DNA.

CRISPR-Cas9 is rapidly evolving as the tool of choice for genome editing in mammalian cells. The delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that limit overall efficiency and general ease of use.

Dr. Adam Ameur talks with Behind the Bench at ESHG 2015 about the experiments he has run on the Ion PII Chip and the data he hasgotten. Dr. Ameur has been using cervical cancer samples with HPV16 in his research and using the Ion PII Chip he has been able to multiplex 24 samples onto one chip.

At ESHG 2015, Andy Felton gives us an overview of the newest innovations coming out of Ion Torrent. He has updates on the LiquidBiopsy Platform, Ion AmpliSeq Panels and Designer, Ion Reporter Software with Rare Allele Detection, Fusion Panel designs, and more. Watch to find out more about everything coming out of Ion Torrent.

After his presentation at ESHG 2015, Damien Luk of Thermo Fisher Scientific, shares a little about the user experience of the Applied Biosystems QuantStudio 3 and 5 Real time PCR systems.

The QuantStudio 3 and 5 systems have the same user interface as the rest of the QuantStudio instruments which creates a unified experience for the user and adds the utility of being connected to the Thermo Fisher Cloud. This allows Cloud analysis for numerous applications and allows access to your data from any web-browser.

Mike Lelivelt of Thermo Fisher Scientific gave a great talk during his session

at ESHG and he was kind enough to offer more detailabout the latest advancements coming out of the Sanger Sequencing Platforms. These include a Minor Variant Finder tool, NGS Confirmation and Changes to Data Collection Software that allow the user more flexibility.

Go Digital PCR: Pt 6 – Emerging Analyses is the final segment in our six-part series introducing you to digital PCR (dPCR) and now, how it aids absolute quantitation and rare sequence detection in lesser used applications.

In this interview Dr. Antonio Alonso (National Institute of Toxicology and Forensic Sciences, Madrid Spain) describes the six-dye, 24-locus GlobalFiler™ Kits with five times the discriminatory power, and its utility in differentiating DNA mixt

Last year over 8,000 attendees came to Boston for the American Society of Microbiology scientific conference and likely even more will come as we gather in New Orleans next week. We will be there to answer your questions all while showcasing some of the smartest laboratory solutions available and presenting a great workshop. Let’s get into the details so you can start planning your time in N’Orleans.

Go Digital PCR: Pt 5 – Rare Mutations is the fifth segment in our six-part series introducing you to digital PCR (dPCR) and how it is capable of detecting and quantifying rare mutations for low-prevalence targets in cancer research samples.

Rare mutation detection is a powerful tool in cancer research

In oncologic research, rare mutation detection among a large population of wild type cells poses a significant challenge. The accumulation of mutations in oncogenes or tumor suppressor genes is an important aspect of tumor formation.

We caught up with Stefano Vernarecci from the Forensics Genetics Laboratory of Rome at the Madrid Human Identification Solutions Conference (HIDS) to learn how does the Italian National Police use QuantiFiler™ Trio in their casework. Stefano was keen to share the value of the Degradation Index feature in the Trio that can help save time, money & DNA while processing challenging casework samples.

Very good databases for reference samples & highly sensitive Y-STR chemistries such as Applied Biosystem YFiler® Plus could help close many sexual assault cases.

Dr. Lutz Roewer explains that we must avoid closing a case just because we do not see anything on the autosomal analysis since a dual workflow, using both autosomal and Y-chromosome analysis can help provide full information.

Reading about research is one thing, but nothing beats seeing it for yourself in person. So when we had the opportunity to with meet the people behind the Cheetah Conservation Fund (CCF) and learn more about how sequencing is impacting their conservation efforts, we jumped at it – and how often do you get a chance to see a real Cheetah up-close?

It’s no secret that the cheetah has suffered tragic setbacks at the hands of human-wildlife conflict, and according to CCF, their population declined by 90% in the 20th century.

Very good databases for reference samples & highly sensitive Y-STR chemistries such as Applied Biosystem YFiler® Plus could help close many sexual assault cases.

Dr. Lutz Roewer explains that we must avoid closing a case just because we do not see anything on the autosomal analysis since a dual workflow, using both autosomal and Y-chromosome analysis can help provide full information.

At the Association for Biomolecular Resource Facilities conference in Saint Louis Missouri USA, Mike Lelivelt described the Thermo Fisher TM Cloud analysis capability with not only real-time PCR data but also Sanger Capillary Electrophoresis sequencing data.

Mike Lelivelt (Thermo Fisher Scientific, San Francisco California USA) shared a talk entitled “Integrating the Analysis of Nucleic Acid Data across Multiple Platforms on the Cloud”. We caught up with Mike to discuss his presentation and what the Thermo Fisher TM Cloud has to offer.

Part 3 of our series on Sanger Sequencing by Capillary Electrophoresis is about primer design. Primer design for Sanger sequencing is a very straightforward process with online tools such as the Primer Designer™ Tool which offers >300,000 primer pairs targeting the human Exome as well as the OligoPerfect™ Designer But what about primer design for more specialized Sanger CE applications, such as for human identification?

I interviewed Julio Mulero, a senior staff scientist with Thermo Fisher Scientific whose group develops highly-multiplexed products for human identification, such as the new GlobalFiler® STR kits that amplifies 24 loci simultaneously.